Sains Malaysiana 38(3): 429-434(2009)

Purification of Enzyme and Influence of Substrate Specificity on β-1,6-Glucanase

Gene Expression in Trichoderma harzianum

(Penulenan enzim dan pengaruh pengkhususan substrat ke atas pemaparan

gen β-1,6-glukanase pada Trichoderma harzianum)

M.Muskhazli¹*, G.L.F.Wallis², J.F. Peberdy², H.A.B. Salifah¹, S.N.Nurul¹, G. Rusea¹ & A.A. Nor Azwady¹

¹Department of Biology, Faculty of Science, Universiti Putra Malaysia

43400 UPM Serdang, Selangor D.E. , Malaysia

²School of Biological Studies, University of Nottingham

NG7 2RD Nottingham, United Kingdom

Received: 16 June 2008 / Accepted: 29 August 2008

ABSTRACT

ß-1,6-gluc ana se produced by Trichoderma harzianum has been proven as one of the prime compounds to be excreted onto the hyphae of the pathogen causing localised cell wall lysis at the point of interaction.  This study was conducted in the interest to investigate the regulation of ß-1,6-gluc ana se gene expression in T. harzianum strain BIO10671.  β-1,6-gluc ana se enzyme from the culture filtrate of T. harzianum was purified through precipitation with 80% acetone followed by anion-exchange chromatography and chromatofocusing using Neobar AQ and Mono P HR 5/20 columns, respectively.  Two β-1,6-gluc ana se bands at 32 kDa and 43 kDa in size has been purified. However, four restriction endonucleases digestion revealed only a single copy of ß-1,6-gluc ana se gene was encoded for both ß-1,6-gluc ana se iso zymes.  Fungal cell walls were able to trigger high level expression of gene encoding ß-1,6-gluc ana se.  The expression of ß-1,6-gluc ana se gene was strongly affected by substrate specificity; where the presence of glucose or non ß-1,6-glucan linked substrate will significantly suppress the gene transcriptions.  In spite of this, 24 hours were required for the gene transcription to achieve maximum total mRNA. 

Keywords:  ß-1,6-gluc ana se; Gene; mRNA; purification; substrate specificity

ABSTRAK

ß-1,6-gluc ana se yang dihasilkan oleh Trichoderma harzianum telah terbukti sebagai salah satu daripada komponan utama yang dirembeskan ke dalam hifa patogen dan mengakibatkan hakisan setempat dinding sel di titik sentuhan.  Kajian ini dijalankan dengan tujuan menyelidik kawalaturan pemaparan gen ß-1,6-gluk ana se di dalam T. harzianum strain BIO10671.  Enzim β-1,6-gluk ana s daripada cecair kultur T. harzianum telah ditulenkan menerusi pemendapan dengan 80% asiton diikuti oleh kromatografi penukaran ion dan kromatografi pemfokusan, masing-masing menggunakan kolum Neobar AQ dan Mono P HR 5/20.  Dua jalur β-1,6-gluk ana se pada saiz 32 kDa dan 43 kDa telah ditulenkan. Walau bagaim ana pun pemcernaan empat endonukleus penyekat memaparkan hanya satu salinan gen ß-1,6-gluk ana se yang mengkodkan kedua-dua ß-1,6-gluk ana se iso zim.  Dinding sel kulat berupaya mencetuskan tahap pemaparan gen ß-1,6-gluk ana se yang tinggi. Pemaparan gen ß-1,6-gluk ana s amat dipengaruhi oleh pengkhususan substrat; kehadiran glukosa atau substrat yang tidak mempunyai jalinan ß-1,6-glukan akan menghalang secara berkesan penyalinan gen.  Namun begitu, tempoh 24 jam masih diperlukan bagi penyalinan gen untuk mencapai jumlah maksima mRNA. 

Kata kunci:  ß-1,6-gluk ana se; Gen; Penulenan, mRNA; Pengkhususan substrat

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*Corresponding author; email: muskhazli@science.upm.edu.my